PROJECT SUMMARY ? PROJECT 4 (UT SOUTHWESTERN AND MOUNT SINAI) Over the past four years, Project 4 has made robust progress in defining gene expression and associated chromatin abnormalities that occur in specific limbic brain regions of depressed humans. Working with Projects 1 through 3, we validated numerous findings from mouse models in human depression, which ensures that the other Projects remain focused on mechanisms relevant to the human syndrome. In parallel, we defined novel transcriptional and epigenetic abnormalities in the depressed human brain. A major milestone is completing RNA-seq of six brain regions from ~100 subjects?half depressed, half control; half male, half female. While the data revealed many genes similarly affected in depressed men and depressed women, striking sex differences were observed as well, suggesting that depression may be a fundamentally distinct syndrome in the two sexes. This dataset helped define the current focus of the other Projects. It also provides the foundation for our proposed experiments. We will extend RNA-seq analysis to an additional cohort of 100 subjects, which is necessary given the heterogeneity of the depression syndrome. We will complement this work with ChIP-seq and, with Projects 2 and 3 by mapping the 3D genome and nucleosome turnover, to begin to define genome-wide the epigenetic mechanisms controlling the aberrant gene expression seen in human depression. We will focus on key target genes identified in these studies as being altered in a sex-specific manner in either prefrontal cortex (PFC) or nucleus accumbens (NAc) in human depression, regulation since replicated in mouse models. Among the regulated genes are those that encode several long-noncoding RNAs for which homologues do not exist in rodents?emphasizing the importance of gene discovery in human brain. As well, we will relate gene and chromatin changes to demographic features of the cases and controls, examining the effect of early life stress and moving beyond syndromal depression to key domains of behavioral abnormalities. We also will continue to build our bank of depressed and control human brains. The work of Project 4 thus embodies the bidirectional translation that defines this Center and its promise of discovering new mechanisms for the pathophysiology of depression and other stress-related illnesses.